COTI-2 | Reactivating Mutant p53
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COTI-2 | Reactivating Mutant p53

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Presentation given by Dr. Wayne Danter at the 16th International p53 Workshop in Stockholm Sweden on June 16, 2014. This presentation provides an overview of Critical Outcome Technologies' lead cancer ...

Presentation given by Dr. Wayne Danter at the 16th International p53 Workshop in Stockholm Sweden on June 16, 2014. This presentation provides an overview of Critical Outcome Technologies' lead cancer drug candidate, COTI-2, and outlines its p53-dependent mechanism of action.

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COTI-2 | Reactivating Mutant p53 Presentation Transcript

  • 1. Stockholm, Sweden | June 2014 COTI-2 | Reactivating Mutant P53
  • 2. FORWARD LOOKING STATEMENTS When used anywhere in this presentation, the words expects, believes, anticipates, estimates, and similar expressions are intended to identify forward-looking statements. Forward-looking statements herein may include statements addressing future financial and operating results for Critical Outcome Technologies Inc., (COTI). COTI has based these forward-looking statements on its current expectations about future events. Such statements are subject to risks and uncertainties including, but not limited to, the successful implementation of COTI’s strategic plans, the acceptance of new products, the obsolescence of existing products, the resolution of existing and potential future patent issues, additional competition, changes in economic conditions, and other risks described in documents COTI has filed with the Toronto Stock Exchange and Ontario Securities Commission. All forward-looking statements in this document are qualified entirely by the cautionary statements included in this document and such filings. These risks and uncertainties could cause actual results to differ materially from results expressed or implied by forward-looking statements contained in this document. These forward-looking statements speak only as of the date of this document. 2
  • 3. 3 COTI-2 INTRODUCTION • COTI-2 is a small molecule with a novel mechanism of action, discovered by our CHEMSAS process – Originally thought to be an AKT modulator/inhibitor • It is a 3rd generation thiosemicarbazone engineered for low toxicity and easily synthesized in 3 steps • It demonstrates strong in vitro and in vivo efficacy • COTI-2 is effective in cancer cells/tumors with many common p53 mutations N N N H S N N N 3rd Generation Thiosemicarbazone
  • 4. 4 COTI-2 IC50 STRONGLY CORRELATED WITH p53 STATUS • TP53 is the most frequently mutated gene in human cancer with mutation frequencies ranging from 38% to 96%1 – Most frequent mutations occur in 6 “hotspot” codons2 • 23 cell line were evaluated for the relationship between their COTI-2 IC50 and p53 “hotspot” mutational status (amino acid residues 175, 248, and 273) – COTI-2’s efficacy was strongly associated with the presence of p53 “hotspot” mutations (Fisher exact, p = 0.0045) 1Freed-Pastor WA & Prives C (2012) Genes Dev. 26: 1268-1286. 2Yu X et al (2012) Cancer Cell, 21: 614-625.. LOW IC50 (< 250 nM) HIGH IC50 (≥ 250 nM) TOTAL Hotspot p53 Mutations (175, 248, 273) 13 0 13 WT p53 5* 5 10 TOTAL 18 5 23 Common p53 Gene Mutations COTI-2 Efficacy Associated with p53 Hotspot Mutations ~28% of human cancers * These cell lines also have mutations in p73 or the PI3K/AKT/mTOR pathway
  • 5. -10.00 -9.00 -8.00 -7.00 -6.00 -5.00 -4.00 -3.00 5 COTI-2 IC50 DISTRIBUTION IN HUMAN CANCER CELL LINES • COTI-2 IC50 distribution in various human cancer cell lines as illustrated by a waterfall chart shows 3 major groups of cell lines Normal Human WBCs MCF7 and NCI-H460: WT p53 + PIK3CA mutation U87MG: WT p53 + PTEN mutation NCI-H292: WT p53 + p73 mutation HCT116: WT p53 + PIK3CA mutation PANC-1: p53 (R273H) OVCAR-3 PK/PD xenograft p53 (R248G) GROUP 1 GROUP 2 GROUP 3 p53 = Wild-type Mutants = p53 Wild-type AND p73, PI3K, or PTEN Mutant p53 = Null Mutants p53 = Hotspot & Non-Hotspot Mutants
  • 6. 6 COTI-2 INDUCES A CONFORMATIONAL CHANGE IN MUTANT P53 • COTI-2 induces a ‘wild-type-like’ conformational change in mutant p53R175H in TOV- 112D ovarian cancer cell line • Data also suggests a mutant p53 conformational change also occurs in PANC-1 (p53R273H) pancreatic and OVCAR-3 (p53R248Q) ovarian cancer cell lines Experimental Design • Fluorescently labeled antibodies specific for wild-type & mutant p53 used to assess cells grown in the presence/absence of COTI-2 • PAB1620-stained cells show wild-type p53 and PAB240-stained cells show mutant p53 • Cy3 refers to the fluorescent signal emitted by the fluor attached to the antibodies • DAPI-staining indicates intact nuclei and provides the assurance that the cells are intact
  • 7. 7 COTI-2 HAS NO AFFECT ON P53 WILD-TYPE CONFORMATIONAL STATUS • COTI-2 has no affect on the wild-type p53 conformation as demonstrated by the H460 NSCLC cell line, which was used as a control Experimental Design • Fluorescently labeled antibodies specific for wild-type & mutant p53 used to assess cells grown in the presence/absence of COTI-2 • PAB1620-stained cells show wild-type p53 and PAB240-stained cells show mutant p53 • Cy3 refers to the fluorescent signal emitted by the fluor attached to the antibodies • DAPI-staining indicates intact nuclei and provides the assurance that the cells are intact
  • 8. 8 COTI-2 TARGETS P53 MUTANT PROTEIN • COTI-2 significantly reduces p53 mutant protein levels and significantly increases wild-type p53 protein levels in TOV-112D cells likely by inducing a conformational change • COTI-2 has no significant effect on p53 protein levels in the H460 cell line, which do not carry the mutant p53 protein • (*) Significant difference in p53 protein levels between COTI-2 treated and untreated cells (control) Mutant p53 Levels in the Presence/Absence of COTI-2 Wild-type p53 Levels in the Presence/Absence of COTI-2 0 20 40 60 80 100 120 TOV-112D H460 MeanFluorescenceIntensity (ArbitraryUnits) Cell Line Control COTI-2 * 0 20 40 60 80 100 TOV-112D H460 MeanFluorescenceIntensity (ArbitraryUnits) Cell Line Control COTI-2 *
  • 9. 9 COTI-2 AFFECTS A WIDE RANGE OF P53 MUTATIONS • COTI-2 inhibited a wide range of p53 mutations at concentrations  1.0 M – Fifteen p53 mutations were inhibited by COTI-2 (shown below in red) – The p53 mutations correspond to those most frequently occurring in various human cancers Experimental Design • HCT116 p53 (-/-) constructs were transfected with plasmids bearing various p53 mutant genes • Each transfectant was exposed to 1.0 M of COTI-2 for 72 hrs and viability assessed • The cell viability was determined using the CellTiter-Blue® Assay Unlike other p53 targeted drugs in development COTI-2 targets a wider range of p53 mutations N C S G C H L S C R W G Q C G S R Y V D A L P W L H P S F R175 C275R273R248G245Y220
  • 10. 10 COTI-2 REACTIVATES MUTANT P53 • COTI-2 reactivates the tumor suppressor properties of p53 as demonstrated by the induction of p21, which is a p53-dependent gene – COTI-2 induces p21 at 3 and 12 h post-exposure in both the HCT116 p53 (R175H) and (G245C) mutant constructs – No difference in p21 protein levels were observed in the HCT116 p53 (-/-) construct Experimental Design • Cells were incubated with/without COTI-2 and protein extractions performed at 2 and 12 h post-incubation • Protein levels were detected via western blots using p53, p21, and Erk2 specific antibodies 1 2 3 4 HCT116 p53 (R175H) HCT116 p53 (G245C)HCT116 p53 (-/-) 3h - + 12h - + 1 2 3 4 p53 p21 Erk2 COTI-2 3h - + 12h - + 3h - + 12h - + 1 2 3 4
  • 11. COTI-2 REACTIVATES MUTANT P53 THROUGH ZINC CHELATION • 8 genes involved in metal ion chelation up- regulated by COTI-2 (p<0.01) • Iron chelation unlikely to be significant effect • Pre-treatment of p53 mutant cell lines with TPEN markedly reduces COTI-2 effectiveness • COTI-2 increases intracellular zinc concentrations • Experiments are ongoing to better understand the role of zinc in COTI-2 effectiveness 11
  • 12. 12 CONFIRMATION OF P53-DEPENDENT MOA • Gordon Mills, MD., PhD., Chair of the Department of Systems Biology and the Co- Director of the Khalifa Institute for Personalized Cancer Therapy at The University of Texas MD Anderson Cancer Center in Houston, Texas conducted a number of cell-based experiments to further elucidate the activity of COTI-2 on various p53 mutations • Dr. Mills’ research confirmed – COTI-2 is most active in mutant p53 tumors – COTI-2’s activity in many specific p53 mutations is striking – COTI-2’s activity can be augmented by the presence of mutations in the AKT signaling pathway – Results of in vitro/in vivo studies in combination with the potential MOA warrant further clinical development “These results are encouraging given the central importance of p53 gene mutations in many cancers” Dr. Gordon Mills, MD Anderson Cancer Center
  • 13. 13 COTI-2 INDUCED NO SIGNIFICANT RESISTANCE • COTI-2 induced no significant resistance through 5 generations in both cell lines • Both Cisplatin and Placlitaxel induce significant increases in IC50 after first generation of selection compared with COTI-2 • COTI-2 induced a statistically significant decrease in IC50’s (i.e., Collateral Sensitivity) 0 0.5 1 1.5 2 2.5 3 Parental cells Round1 selection Round2 selection Round3 selection Round4 selection COTI-2 COTI-219 Cisplatin Paclitaxel 0 1 2 3 4 5 6 7 8 Parental cells Round1 selection Round2 selection Round3 selection Round4 selection COTI-2 COTI-219 Cisplatin Paclitaxel IC50Ratio DMS153 SCLC SHP77 SCLC IC50Ratio
  • 14. 14 COTI-2 PRODUCED SIGNIFICANT TGI AS A SINGLE AGENT • COTI-2 administered PO produced a significant tumor growth inhibition as a single agent in the OVCAR-3 ovarian cancer tumor model Experimental Design • OVCAR3 human tumor cells inoculated subcutaneously in the right and left flanks of female athymic mice (NIH III nu/nu), allowed to grow up to 75 to 100 mm3 • Groups of 12 mice each were dosed PO with COTI-2 in phosphate citrate buffer (75 or 100 mg/kg) with a schedule of 5X per week until study end • Tumor volumes were graphed as means (SE) and significant difference between groups was determined using Student’s T- test (p<0.05) • (*) Tumors significantly reduced by COTI-2 in all treatment groups relative to vehicle control 0 50 100 150 200 250 0 5 9 16 23 30 37 44 51 61 TumorVolume(mm3) Study Day Group 4 = Vehicle PO Group 5 = COTI-2 75mg/kg PO Group 6 = COTI-2 100mg/kg PO Effect of Treatment on Tumor Volume * * * * * * * * * * * *
  • 15. 15 SUMMARY • COTI-2 is a small molecule with a novel mechanism of action, discovered by our CHEMSAS process • MOA – COTI-2 appears to restore wild-type like functional activity to a wide range of p53 mutants through zinc chelation • In vitro efficacy – COTI-2 demonstrates nanomolar activity in multiple human cancer cell lines – COTI-2 did not induce cross-resistance compared to cisplatin and paclitaxel • In vivo efficacy – When administered PO, COTI-2 induced significant tumor growth inhibition (85.5%) in an ovarian tumor xenograft model without any overt toxicity in mice • Clinical development strategy – IND filing is planned for mid-2014 – Gynecological (ovarian, endometrial, and cervical) cancer patients failing 1st line therapy • FDA Orphan status granted June, 2014 “for the treatment of ovarian cancer”
  • 16. 16 COTI-2 NEGATIVELY MODULATES THE PI3K/AKT/MTOR PATHWAY 1Hennessy BT et al (2005) Nature Rev Drug Discov, 4: 988-1004. • This pathway has been implicated in cancer and is involved in cell proliferation, survival, motility and morphology1 • COTI-2 negatively modulates the PI3K/AKT/mTOR pathway – COTI-2 directly inhibits PI3K via the upregulation of PIK3IP1 – Increased/restored p53 function stimulates PTEN activity – Increased/restored p53 function leads to AKT protein degradation PI3K AKT MDM2 p53 FOXO1 BAD GSK3B mTOR S6KCASP-9 RTK Apoptosis Cell cycle regulation DNA repair Apoptosis Cell cycle regulation Growth Translation PIK3IP1 PTEN COTI-2 Upregulates PIK3IP1 Study # COTI-07-101
  • 17. Follow @CriticalOutcome on Twitter COTI-2 | Reactivating Mutant P53